(CANCER RESEARCH 55, 2615-2619, June 15, 1995]

Inhibition of the Growth of Various Human and Mouse Tumor Cellsby l,15-Bis(ethylamino)-4,8,12-triazapentadecane1

Kazuei Igarashi,2 Kunihiko Koga, Yong He, Tomomi Shimogori, Hisao Ekimoto, Keiko Kashiwagi, and

Akira ShirahataFaculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, lnage-ku, Chiba 263 [K. L, Y. H., T. S., K. Ka.¡;Research Laboratories, Pharmaceutical Group, NipponKti\aku Co., Ltd., 3-12 Shimo, 3-Chome, Kita-ku, Tok\o 115 [K. Ko., H. £./; and Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado 350-02 [A. S.J,

Japan

ABSTRACT

Effects of l,15-bis(ethylamino)-4,8,12-triazapentadecane (BE3333), the

least toxic bis(ethyl)pentaamine, on the growth of tumor cells were studiedin in vitro systems and with tumor xenografts in mice. BE3333 suppressedornithine decarboxylase and S-adenosylmethionine decarboxylase, induced spermidine/spermine N'-acetyltransferase, and thus decreased the

amount of polyamines. BE3333 accumulated in cells at a concentration3-5-fold that of spermine in control cells through the polyamine transport

system. The accumulated BE3333 inhibited protein synthesis, especiallymitochondria! protein synthesis, and decreased the amount of ATP. Theinhibition of protein synthesis was correlated with the subsequent inhibition of cell growth. BE3333 showed inhibitory effects in in vitro systemsagainst the growth of mouse FM3A mammary carcinoma cells, humanSW480 and SW620 colon tumor cells, Lu-65A and A549 lung tumor cells,MCF-7 breast tumor cells, and MALME-3M and A375 melanoma cells ata range of 0.5-10 U.M.Intravenous (30 mg/kg) or i.p. (50 mg/kg) daily

injections of BE3333 for 5 or 7 days greatly suppressed the growth ofhuman colon tumor SW620 xenotransplanted into nude mice. Similarantitumor activity was obtained with continuous infusion of BE3333 intothe peritoneal cavity (80 mg/kg), but not with p.o. administration (200mg/kg). BE3333 also showed inhibitory effects against the growth of lungtumors (Lu-65, Lx-1, Lc-1, and Lu-61), stomach tumors (Sc-6 and St-15),

and melanoma (SEKI) xenotransplanted into nude mice. The resultsindicate that BE3333 is effective against both rapid- and slow-growingtumors, with reasonable short-term host toxicity.

INTRODUCTION

Since polyamine biosynthesis is closely related to cell growth, thepossibility that inhibitors of polyamine biosynthesis may function asanticancer agents has been examined (1). Two chemicals, used widelyas inhibitors in polyamine biosynthesis, are DFMO,3 an inhibitor of

ODC (2), and MGBG, an inhibitor of SAMDC (3). These inhibitors oftwo different segments of polyamine biosynthesis have exhibitedantitumor activity in experimental tumor studies (4, 5), and the anti-

tumor effect of DFMO has been studied extensively (1). In addition,the combination of DFMO-MGBG has been shown to be more effec

tive therapeutically than either drug alone in a number of in vivomodel systems (6-11). The in vivo usefulness of these inhibitors,

however, has been limited by regulatory homeostatic responsesunique to the polyamine pathway. For example, in our experiments(10), combined treatment with DFMO and MGBG caused a remarkable regrowth after its termination.

Received 12/16/94; accepted 4/17/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by a Grant-in-Aid for Scientific Research from the Ministry

of Education, Science, and Culture (Japan) and by research aid from the ResearchFoundation for Pharmaceutical Sciences.

2 To whom requests for reprints should be addressed.3 The abbreviations used are: DFMO, a-difluoromethylornithine; ODC, ornithine

decarboxylase; MGBG, methylglyoxal bis(guanylhydrazone); SAMDC, S-adenosylmethionine decarboxylase; SSAT, spermidine/spermine /V'-acetyltransferase; BE3333, 1,15-bis(ethylamino)-4,8,12-triazapentadecane; BE4444, 1.19-bis(ethylamino)-5,10,15-triaza-

nonadecane.

As an alternative to specific inhibitors, bis(ethyl)polyamine analogues have been developed as antiproliferative reagents (12, 13).These reagents cannot only negatively regulate the synthesis of ODCand SAMDC (14), but can also induce SSAT activity (15, 16). Theanalogues could deplete intracellular polyamines almost completely,and the level of accumulation of the analogues was nearly equal to theoriginal spermidine and spermine contents (17). Thus, the analogueswere thought to inhibit cell growth through the depletion of polyamines. We found that A'1,A'12-bis(ethyl)spermine can substitute for

the functions of spermine in various aspects and accumulates in cellsat a concentration up to 5-fold that of spermine in control cells (18,

19); we suggested that not only polyamine deficiency but also theaccumulation of /V'.A^-bisiethylJspermine may be involved in the

inhibition of cell growth (19). Recently, it has been reported that theinhibition of cell growth by A^.A^-bisiethy^spermine correlated with

the intracellular accumulation of the analogue (20). We also foundthat the accumulated spermidine and spermine inhibited cell growth(21). The accumulated polyamines and bis(ethyl)polyamine analoguescaused the inhibition of protein synthesis, especially mitochondrialprotein synthesis, leading to a decrease in ATP (21, 22).

Since the inhibition by bis(ethyl)polyamines of cell growth in invitro systems was very potent, the antitumor activity of N\N11-

bis(ethyl)norspermine was examined against human MALME-3 mel

anoma xenografts, and it was found that the analogue fully suppressedgrowth of the melanoma (23, 24). As the degree of inhibition of cellgrowth by bis(ethyl)pentaamines was stronger than that by bis(ethyl)-

tetraamines in an in vitro system (22), we examined the antitumoractivity of BE3333, the least toxic bis(ethyl)pentaamine, against various human tumors xenotransplanted into nude mice, and found thatthe analogue has strong antitumor activities. In this regard, it has beenreported recently that BE4444 also shows strong antitumor activitiesagainst human tumors xenotransplanted into nude mouse (25).

MATERIALS AND METHODS

Materials. BE3333 5HC1 was synthesized by removal of the protectinggroups from benzylated and p-toluenesulfonylated BE3333 prepared by N-alkylation of A'1-,A'4-,/V7-tribenzylnorspermidine with /V-ethyl-A'-(3-bromopro-

pyl)-p-toluenesulfonamide according to the method for the synthesis of pen-taamines (26), crystals from water-ethanol. The structure was confirmed by

elemental analysis. Mouse mammary carcinoma FM3A cells were kindlysupplied by Dr. H. Matsuzaki, Saitama University (Urawa, Japan). HumanSW480 and SW620 cells were obtained from Dainippon Pharmaceutical Co.(Osaka, Japan). A375, MALME-3M, and MCF-7 cells were obtained fromAmerican Type Culture Collection (Rockville, MD). A549 and Lu-65A cells

were supplied by Riken Cell Bank (Tsukuba, Japan) and the Japanese CancerResearch Resources Bank (Tokyo, Japan), respectively. Lc-1, Lu-61, Lu-65,

Lx-1, SEKI, Sc-6, and St-15 cells were obtained from the Central Institute for

Experimental Animals (Kawasaki, Japan).Cell Culture. Cells were grown in an environment of 5% CO2 in humid

ified air. Mouse FM3A mammary carcinoma cells were cultured in ES medium(Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 50units/ml streptomycin, 100 units/ml penicillin G, and 2% heat-inactivated FCS.Lu-65A lung tumor and MCF-7 breast tumor cells were grown in RPMI 1640

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ANTITUMOR EFFECTS OF POLYAMINE ANALOGUES

with 10% PCS and 60 fig/ml kanamycin. A375 melanoma and A549 lungtumor cells were grown in DMEM with 10% PCS and 60 jig/ml kanamycin.MALME-3M melanoma, SW480, and SW620 colon tumor cells were grown inL-15 medium with 10% PCS, 2 mM glutamine, and 60 fig/ml kanamycin.

Tumor Growth and Drug Treatment. Tumor cells (2x2x2 mm3) were

inoculated into BALB/c (nu/nu) mice (approximately 20 g body weight; JapanClea Laboratories, Tokyo, Japan). After inoculation, the tumors were monitored by measuring two perpendicular tumor diameters, width (the smallestdimension) and length (the largest dimension) with calipers. Tumor growthdata were expressed as tumor volumes (mm3) calculated from the followingequation: length x width2 X 0.52 (27). When tumor volumes reached 80 mm3,

BE3333 was daily given either i.V., i.p., or p.o. for 5 days (days 0-4) or for 7

days (days 0-6). Continuous infusion into the peritoneal cavity was also

performed with Alza Model 1007 D micro pumps (Palo Alto, CA) accordingto the method of Siegall et al (28). The statistical significance of differencesin animal weight and tumor volume was assessed by the Student's t test.

Enzyme Assays. FM3A cells (5 X IO7) or tumor cells (50-200 mg) were

suspended in 0.8 ml Buffer A containing 10 mM Tris/HCl (pH 7.5), 1 mM DTT,20% glycerol, 1 mM EDTA, and 20 JJ.M6-amino-2-naphthyl-4-guanidinoben-

zoate dihydrochloride, a strong proteinase inhibitor (29). They were frozen,thawed, and then homogenized with a Teflon homogenizer. The homogenatewas centrifuged for 10 min at 12,000 X g. The supernatant was dialyzedagainst Buffer A and used for the enzyme assays. Assays of ODC, SAMDC,and SSAT were performed as described previously (30, 31) with some modifications (21). Assay for polyamine transport was performed as described(32). The protein concentration was determined according to the method of

Lowry et al. (33).Measurements of Polyamines, BE3333, and ATP. FM3A cells (6 X IO6)

or tumor cells (10-50 mg) were homogenized with 0.3 ml 0.2 M HC1O4 and

centrifuged for 10 min at 12,000 X g. The supernatant thus obtained was usedfor the following assays. Polyamines (putrescine, spermidine, and spermine)were measured as described previously (34). BE3333 was analyzed afterdansylation according to the previous publication (22). ATP was measuredusing the luciferase enzyme system (35) after neutralization with l M KOH

containing 50 mM K2HPO4.Analysis of Total and Mitochondria! Protein Synthesis. PM3A cells

(5 X 10ft) cultured in the presence of 10 JIM BE3333 were incubated for 2 hwith 3.7 MBq [35S]methionine in the presence and absence of emetine (0.2

mg/ml), which inhibits cytoplasmic protein synthesis specifically (36). Thecells cultured in the absence of emetine were harvested, and total proteinsynthesis was measured by counting the radioactivity insoluble in hot trichlo-

roacetic acid. The mitochondrial fraction was obtained from the cells culturedin the presence of emetine by homogenization with a buffer containing 0.25 Msucrose, 1 mM EGTA, and 10 mM HEPES/KOH (pH 7.4) followed by differential centrifugation (36). Mitochondrial protein synthesis was measured bycounting the hot trichloroacetic acid-insoluble radioactivity in the mitochon

drial fraction.

RESULTS

Effect of BE3333 on the Growth of Mouse Mammary Carcinoma FM3A Cells in Culture. In the previous communication (22),the effect of bis(ethyl)pentaamines on the growth of FM3A cells inculture was examined at relatively high concentrations (0.25-0.3

mM). In the present experiments (Fig. 1), the effect of BE3333 on the

—¿�1x10

I

1x10

012345

Time ( days )

Fig. 1. Effect of BE3333 on the growth of mouse FM3A cells. O, control; •¿�,0.3 /O.MBE3333; D, l |U.MBE3333; •¿�,10 fiM BE3333; and A, 100 f¿MBE3333. Each value is theaverage of three determinations. SE was within ±10% for each point.

growth of FM3A cells was examined at concentrations of 0.3-100 JXM.

The inhibition of cell growth by BE3333 was observed to be dosedependent, and IC50 was 0.48 JU.M.Furthermore, BE3333 caused celldeath on day 6.

Polyamine and BE3333 contents and polyamine metabolizing enzymes in FM3A cells treated with 10 /AMBE3333 were measured(Table 1). BE3333 accumulated in cells at a concentration up to 5-fold

that of spermine in control cells. The decrease in polyamine contentsstarted with putrescine followed by decreases in spermidine andspermine. Thus, the decrease in polyamine contents at the initial phaseby the treatment of cells with BE3333 paralleled the decrease in ODCand SAMDC activities. SSAT then increased in correlation with thedecrease in spermine. BE3333 was probably transported by the polyamine uptake system, since it inhibited spermidine uptake with almostthe same efficiency as spermine (Fig. 2). The inhibition of mitochondrial protein synthesis was greater than that of total protein synthesis,and a decrease in ATP content was observed (Fig. 3). Swelling ofmitochondria was also observed by electron microscopy in cellstreated with BE3333 for 2 days (data not shown).

Effect of BE3333 on the Growth of Human Tumor Cells inCulture. IC5()of BE3333 against various human tumor cells (colonadenocarcinoma, small cell lung carcinoma, lung adenocarcinoma,breast carcinoma, and melanoma) in culture was then measured (Table2). The values obtained ranged from approximately 0.5 to 3 JU.M.Therewas a tendency that rapidly growing cells needed higher concentrations of BE3333 to inhibit cell growth. The only exception wasMALME-3M melanoma cells, which grew slowly but the IC50 value

was rather high (1.16 /UM).

Table 1 Polvamine and BE3333 contents and pohamìnemelaholizing enzymes in FM3A cells

CultureControlBE3333(10

(IM)Incubation

time(h)4872612244872Polyamines"

(nmol/mgprotein)Putrescine3.293.101.02<0.1<0.1<0.1<0.1Spermidine13.211.37.842.730.85<0.1<0.1Spermine8.287.638.016.734.252.37<0.1BE333315.622.237.240.839.7Enzymeactivity" (pmol/min/mgprotein)ODC13612742.515.6<1<1<1SAMDC16.514.16.272.500.38<0.1<0.1SSAT79.076.8355796161233883510

' Each value is the average of two determinations.

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ANTITUMOR EFFECTS OF POLYAMINE ANALCXiULS

Effect of BE3333 on the Growth of Human Tumors Xenotrans-

planted into Nude Mice. The antitumor effect of BE3333 was firstexamined with SW620 colon adenocarcinoma xenotransplanted intonude mice (Fig. 4). When BE3333 was given i.v. at a dose of 20-30mg/kg/day for 7 successive days (days 0-6), the growth of tumors

was strongly inhibited (Fig. 4A). However, the growth recoveredgradually after cessation of the administration. When BE3333 wasgiven i.p. at a dose of 50 mg/kg/day, a similar inhibitory effect to thatwith 30 mg/kg/day i.v. was obtained (Fig. 4ß).The ¡.p.administrationof 80 mg/kg/day was toxic, because all mice died by day 5. The i.p.administration of BE3333 at a dose of 30 mg/kg/day had a smallerinhibitory effect than at 50 mg/kg/day. Continuous infusion intothe peritoneal cavity (50 or 80 mg/kg/day) by micro pumps alsoshowed a strong antitumor effect (Fig. 4C). However, there was no

16Inhibitor ( uM )

Fig. 2. Effect of BE3333 and spcrmine on [14C]spermidine uptake. Mouse FM3A

mammary carcinoma cells were used for the assay. One hundred percent activity was 32.4pmol/min/mg protein. O, with BE3333 and •¿�,with spermine. Each value is the averageof duplicate determinations.

30

Fig. 4. Antitumor activity of BE3333 against SW620 colon adenocarcinoma xenotransplanted into nude mice. BE3333 was administered for 7 days from days 0 to 6. A, i.v.injection. O, control; •¿�.10 mg/kg/day; •¿�.20 mg/kg/day; and A, 30 mg/kg/day. B, i.p.injection. O, control; •¿�,50 mg/kg/day; and •¿�80 mg/kg/day. C, continuous infusion intothe peritoneal cavity. O, control; •¿�,50 mg/kg/day; and •¿�.80 mg/kg/day. D, p.o.administration. O, control; •¿�.150 mg/kg/day; and •¿�2(K)mg/kg/day. Vertical (A, K, andC) and horizontal (C) arrows, injection days. Each group consisted of four mice. Eachsymbol with its vertical bar, mean and SD, respectively.

100

50

12 24 36

Time (h)

48

Fig. 3. Effect of BE3333 on total and mitochondrial protein synthesis and ATPconcentration. Total (O) and mitochondrial (•)protein synthesis and ATP content (•)were measured as described in "Materials and Methods." Maximum values (100%) fortotal and mitochondrial protein synthesis and ATP content were 1.12 X IO7 and6.16 X 10? cpm [^S]methionine incorporated/mg protein and 12.2 nmol/mg protein,

respectively. Each value is the average of duplicate determinations.

Table 2 Growth inhibitor*' activities of BE3333 against human tumor cell lines

TumorcellsSW480

(colon)SW620(colon, lymphnodemetastasis)Lu-65A(lung carcinoma, smallcell)A549

(lungadenocarcinoma)MCF-7(breast)MALME-3M(melanoma)A375

(melanoma)Doubling

time(h)"39.031.221.343.020.1145.020.0IC50(MM)"0.600.542.010.492.981.161.50

" Each value is the average of three determinations.

effect when BE3333 was given p.o. at a dose of 150-200 mg/kg/day

(Fig. 4D).Weight loss was observed with i.p. (Fig. 5, B and C) but not with

i.v. injection (Fig. 5A). The greatest weight loss (12%) with ¡.p.injection (50 mg/kg/day) was observed on day 6, while that (24%)with continuous infusion into the peritoneal cavity (80 mg/kg/day)was observed on day 12.

Then the effects of BE3333 on the growth of various human tumors(SW620 colon adenocarcinoma, Lu-65 large cell lung carcinoma,Lx-1 small cell lung carcinoma, Lc-1 and Lu-61 squamous cell lungcarcinoma, Sc-6 and St-15 gastric tumor, and SEKI melanoma)xenotransplanted into nude mice were examined by a 30-mg/kg/dayi.v. injection or 50-mg/kg/day i.p. injection for 5 successive days. The

tumor growth was followed for 26 days, and tumor volumes and bodyweight on the most effective day are shown in Table 3. BE3333 waseffective against all tumors. The maximum effect was obtained withLc-1 squamous cell lung carcinoma, and the minimum with St-15

gastric tumor. Weight loss was more severe with i.p. than with i.v.treatment, with the maximum (20%) being observed in mice bearingLu-65 large cell lung carcinoma.

Polyamine and BE3333 contents and polyamine metabolizing enzymes in SW620 and Lu-65 tumor cells xenotransplanted into nude

mice were then measured on days 5 and 15 (Table 4). BE3333 (30mg/kg/day) was given i.v. for 5 successive days (days 0-4). BE3333had accumulated in tumor cells at an amount about 3-fold that of

spermine in control tumor cells on day 5. The decrease in polyaminecontent was significant, paralleling the decreases in ODC andSAMDC activities and the increase in SSAT activity. However, theactivities of ODC and SAMDC were very low compared with those in

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ANTITUMOR EFFECTS OF POLYAMINE ANALOGUES

Fig. 5. Change of body weight of nude mice carrying SW620 colon adenocarcinomabyBE3333. Nude mice were treated as described in the legend to Fig. 4.

cultured FM3A cells (Table 1). On day 15, the amount of accumulatedBE3333 had decreased in conjunction with the recovery of cell growthand the increase in polyamine content.

DISCUSSION

It has been reported that bis(ethyl)norspermine has antitumor activity against human melanoma xenografts (23, 24), and that BE4444has antitumor activity against several human tumor xenografts (25).However, those bis(ethyl)polyamine derivatives are effective againstrelatively slow-growing tumors. In this study, we examined the effect

of another derivative, BE3333, on the growth of various humantumors xenotransplanted into nude mice, and found that it is effectiveagainst both rapid- and slow-growing tumors. The i.v. administration

(60 mg/kg) of bis(ethyl)norspermine was not effective against SW620colon adenocarcinoma and Lu-65 large cell lung carcinoma under our

experimental conditions (data not shown). As for BE4444, largeamounts could not be administered because of its toxicity, precludingany assessment of its possible effect against aggressive tumors.

As for the mechanism of the antitumor activity of bis(ethyl)nor-spermine, it has been reported that SSAT-induced polyamine deple

tion plays a determining role in at least the initial phase of the

antitumor response (24). However, a direct correlation between SSATinduction and growth inhibition does not always exist for all cell linesand analogues (37). We recently reported with in vitro experimentsthat the inhibition of cell growth by bis(ethyl)polyamine analoguescorrelated with their intracellular accumulation, and that the accumulation caused the inhibition of protein synthesis, especially mitochondria! protein synthesis, and then a decrease in ATP content (21, 22).In the present study, we showed that BE3333 accumulated in thetumor cells xenotransplanted into nude mice at an amount about3-fold that of spermine in control cells (Table 4). However, SSAT

only showed a small increase. Taken together, our results stronglysuggest that the antitumor activity of BE3333 is mainly due to theinhibition of protein synthesis, especially of mitochondria! proteinsynthesis. Still, the possibility that BE3333 may also be effective dueto its multiple actions cannot be ruled out.

Although we obtained similar antitumor activity with i.p. (50 mg/kg) and i.v. (30 mg/kg) injections of BE3333, it should be noted thatweight loss was more severe with the former. The rapid diffusion ofBE3333 by i.v. injection may decrease the toxic effects of the drug.However, this is difficult to state conclusively because of differences

Table 3 Antitumor effect of BE3333 on human tumor cells \enotransplantedinto nude mice

Tumorcells"SW620Lu-65Lx-1Lc-1Lu-61Sc-6St-15SEKIcont/i.v.cont.i.p.cont.i.v.i.p.cont.i.V.i.p.cont.i.v.cont.i.v.cont.i.V.i.p.cont.i.v.i.p.cont.i.V.i.p.Day*779966611111111111010101010101010888nc43534435545J65444455545Bodyweight'

(g)21.5

±1.220.2±1.120.7+1.119.3

±0.121.1

+2.418.9±1.517.9

±0.417.2

±0.518.0±1.816.9

±1.421.1

±2.520.8±0.922.6

+1.420.8±1.019.6

±1.218.3±1.618.2

±1.319.8

±1.320.1±1.218.8±3.816.0

±0.716.4±1.015.2

±2.0P"0.10920.07280.08670.03790.21740.32020.42210.01690.12410.08360.36150.31770.28820.2055Tumor

volume(mm3)303.8

±42.387.8±47.7158.6±82.553.1

±1.5334.3

±178.7110.9±66.870.9

±39.2411.6

+83.9209.6±112.793.8

+44.11058.5

±679.5145.9±51.3250.7

±145.683.9±24.01030.3

+228.1320.3±257.8222.1±105.2310.0

±65.1192.0±54.5150.1

±44.7694.0±259.7293.9

±108.1205.4+ 80.6f0.00070.02300.02880.02890.00620.00010.02010.01360.00310.00030.01040.00160.01220.0080

" BE3333 was given i.v. (30 mg/kg) or i.p. (50 mg/kg) for 5 successive days.h Most effective day.' Number of mice in each group.J Data represent mean ±SD.' Student's t test,

cont., control.

Table 4 Polyamine and BE3333 contents and polyamine metabolizing enzymesin human tumor cells xenotransplanted into nude mice

BE3333"Tumor

(30mg/kg)SW620++Lu-65++Time*(day)55155515Polyamines

(nmol/mgprotein)nc555555Putrescine9.96

±0.544.32±0.4512.8+0.412.61

+0.121.40±0.042.98

±0.40Spermidine10.8

+0.481.46±0.0710.7

±0.4013.3

+0.263.42+0.169.28±0.88Spern10.2

+3.07±10.0±7.11

+2.40±4.97

±line0.581.340.530.160.190.21BE333331.27.5024.96.72±3.11+

0.74±0.84±0.27Enzyme

activity(pmol/min/mgroteinODC9.81

+1.152.58±0.245.60±0.406.09

±0.450.85±0.043.12+ 0.07SAMDC1.82

±0.150.82±0.441.73+0.331.93

±0.230.34±0.142.21±0.08SSAT13829013391.12722212175.83815510

" i.p. injection for 5 days.

Day of tumor removal.* Number of mice in each group.(/Data represent mean ±SD.

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ANTITUMOR EFFECTS OF POLYAMINE ANALOGUES

in dosages and routes for delivery. When BE3333 was given p.o., itshowed no antitumor effect at all, reflecting its insufficient absorptionfrom the intestinal tract.

In the case of the antitumor activity caused by the polyaminedeficiency of cells, such as with the combined therapy of DFMO andMGBG (6-11), the activity is essentially cytostatic, but not cytocidal

(38, 39). Thus, marked acceleration of tumor growth followed thecessation of therapy (10). The antitumor activity caused by bis(eth-

yl)polyamine analogues should be cytocidal judging from the experimental results obtained from cell culture of mouse FM3A mammarycarcinoma cells (18). However, tumor growth recovered after cessation of the drug treatment. Further studies will be necessary to clarifywhether BE3333 possesses cytocidal action in vivo.

ACKNOWLEDGMENTS

We thank Dr. K. Samejima for his encouragement during the course of thisstudy and Dr. A. Tomura for his kind help in preparing the manuscript. Thanksare also due to Dr. H. Matsuzaki for his kind supply of mouse mammarycarcinoma FM3A cell lines.

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1995;55:2615-2619. Cancer Res   Kazuei Igarashi, Kunihiko Koga, Yong He, et al.   Cells by 1,15-Bis(ethylamino)-4,8,12-triazapentadecaneInhibition of the Growth of Various Human and Mouse Tumor

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